Discovery of metabolic regulators of DNA topology and cellular responses to topoisomerase II inhibition

项目摘要/摘要 我的研究目的是了解调节拓扑异构酶II(Topo II)的细胞机制。 起作用并对Topo II抑制作出反应。第二类拓扑异构酶，如拓扑异构酶II，是 管理DNA超螺旋结构和解链缠绕的DNA链以促进关键过程，如 如转录、DNA复制和染色体分离1。就人类健康而言，TOPO II是一种 非常重要的癌症靶点，以及临床上使用的各种化疗药物 Topo II产生DNA损伤并杀死复制细胞的能力。生化和结构研究 提供了有关TOPO II的“链通道”机制以及合成的 抑制剂会影响这种活性。然而，该领域缺乏对Topo II活动如何受到监管的明确理解 根据单元格需求。 为了更好地理解Topo II是如何与调控通路和DNA损伤反应联系在一起的，我的目标是 利用酿酒酵母模型系统探索细胞代谢在调节Topo II功能和 对参与Topo II抑制反应的基因进行无偏筛选。在分析 真核生物Topo II的进化保守性，我发现了Topo II上一个高度保守的相互作用的口袋 使用ICRF-187，一种临床批准的用于化疗方案的合成抑制剂2。我后来 发现这个网站也使用白藜芦醇，这是一种在红葡萄酒中发现的天然产品3。这些观察结果 提示这个位点可能是一个孤立的变构位点，并启发了小分子 代谢产物可能以变构方式调节Topo II的功能。初步数据表明，酵母的成分 代谢产物提取物能够调节Topo II的活性。目标1概述了天然产物的纯化 策略和基于质谱学的代谢物配体筛选我将用来鉴定和表征 可能具有TOPO II调节功能的内源性小分子。除了之前的探测之外 细胞新陈代谢和DNA拓扑之间的未知联系，我的目标是揭示参与 细胞对TOPO II异常活性的反应。因此，在目标2中，我提出了一种无偏见的药物遗传学筛选 通过酵母缺失和低晶型文库来识别在临床上存在的影响生长的等位基因- 相关的Topo II抑制剂。总而言之，这些目标有可能发现拓朴之间的新交互作用。 II和调控途径。这样的发现反过来又可能揭开变构调控TOPO II的新方法 抗拓扑异构酶抑制剂联合治疗的作用及新靶点 现有治疗方法的有效性和/或减毒作用。

Project Summary/Abstract The goal of my research is to understand the cellular mechanisms that regulate topoisomerase II (topo II) function and respond to topo II inhibition. Type II topoisomerases, such as topo II, are essential enzymes that manage DNA superhelical structure and decatenate entangled DNA strands to facilitate critical processes such as transcription, DNA replication, and chromosome segregation1. With respect to human health, topo II is a tremendously important cancer target, and a variety of chemotherapeutics are clinically used to exploit the ability to topo II to generate DNA damage and kill replicating cells. Biochemical and structural studies have provided valuable details about the “strand passage” mechanism of topo II and the means by which synthetic inhibitors affect this activity. However, the field lacks a clear understanding of how topo II activity is regulated in accordance with cell needs. To better understand how topo II is connected to regulatory pathways and DNA damage responses, I aim to use the S. cerevisiae model system to explore the role of cellular metabolism in regulating topo II function and perform an unbiased screen for genes involved in responding to topo II inhibition. While analyzing the evolutionary conservation of eukaryotic topo IIs, I discovered a highly conserved pocket on topo II that interacts with ICRF-187, a clinically approved, synthetic inhibitor that is used in chemotherapeutic regimens2. I later discovered that this site also engages resveratrol, a natural product found in red wine3. These observations suggest that this site may be an orphan allosteric site and inspired the hypothesis that small-molecule metabolites may allosterically regulate topo II function. Preliminary data indicate that components of yeast metabolite extracts are able to modulate topo II activity. Aim 1 outlines the natural product purification strategies and mass spectrometry-based metabolite ligand screening I will employ to identify and characterize endogenous small molecules that may have topo II-regulatory function. In addition to probing previously unexplored connections between cellular metabolism and DNA topology, I aim to uncover pathways involved in cellular responses to abnormal topo II activity. Thus, in Aim 2 I propose an unbiased pharmacogenetic screen with yeast deletion and hypomorph libraries to identify alleles that affect growth in the presence of clinically- relevant topo II inhibitors. Together, these aims have the potential to discover novel interactions between topo II and regulatory pathways. Such discoveries, in turn, could unveil new ways to allosterically modulate topo II function and new targets for combinatorial therapy in conjunction with anti-topoisomerase inhibitors to improve the efficacy and/or decrease toxicity of currently available treatments.